Ubiquitin variant modulators of scf e3 ligases and their uses

ABSTRACT

The invention provides ubiquitin variants that specifically bind to SCF E3 ligases, and use of these variants to modulate the activity of SCF E3 ligases.

FIELD OF THE INVENTION

This invention relates to ubiquitin variants that specifically bind toSCF E3 ligases and use of these variants to modulate the activity of SCFE3 ligases.

BACKGROUND OF THE INVENTION

The ubiquitin proteasome system (UPS) plays a central role in proteinhomeostasis through ubiquitination and degradation of substrateproteins. General inhibitors of the proteasome have proven effective incancer therapy (Weathington, 2014), and thus there is great interest indeveloping specific inhibitors of UPS enzymes to explore theirbiological functions and to provide paths to more specific therapeutics.The central player in the UPS is ubiquitin (Ub), a highly conserved76-residue protein. Ub is covalently attached to protein substratesthrough sequential action of ubiquitin activating (E1), ubiquitinconjugating (E2), and ubiquitin ligating (E3) enzymes. E3 ligases bindprotein substrates and thus dictate specificity of ubiquitination.

E3 ligases constitute the largest class of UPS enzymes, with more than600 members encoded by the human genome, and are divided into two majorclasses: a small, well-characterized class of approximately 30 HECT E3ligases and a much larger, but less-characterized class of hundreds ofRING E3 ligases and structurally related variants (Bhowmick). HECT E3ligases form transient thioester linkages with Ub before transferring itto substrates, while RING ligases serve as adaptors to recruitUb-charged E2 enzymes to substrates for Ub transfer. The archetype forthe RING class is the multi-subunit Skp1-Cul1-F-box (SCF) complexfamily, which contains 69 members in humans (Jin, 2004). The SCF enzymecomplexes are composed of constant Rbx1, Cul1, and Skp1 subunits and avariable F-box protein that binds substrates and dictates specificity(FIG. 1 A). Rbx1, the RING protein that recruits the E2 enzyme, bindsthe scaffold protein Cul1, which in turn binds Skp1, an adaptor forF-box proteins. F-box proteins are variable in domain composition butshare a common F-box domain that binds Skp1. F-box proteins aresubdivided into three subfamilies based on the structure of theirsubstrate binding domains including WD40, LRR, and other domains,referred to as the Fbw, Fbl, and Fbo subfamilies, respectively (Jin,2004). Numerous F-box proteins are involved in processes relevant totumorigenesis, including cell proliferation, cell cycle progression, andapoptosis, suggesting that these proteins may be targets for cancertreatment (Wang, 2014).

SUMMARY OF THE INVENTION

In a first aspect, the invention provides ubiquitin variant (Ubv)polypeptides including one or more amino acid substitution in one ormore region of a ubiquitin polypeptide, wherein the region is selectedfrom the group consisting of:

(a) region 1 (amino acids 2-14 of SEQ ID NO:1) wherein the polypeptidecomprises the structure:

X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄

whereinX₂ is selected from the group consisting of H and Q;X₄ is selected from the group consisting of F and L;X₈ is selected from the group consisting of G and L;X₉ is selected from the group consisting of A, S, and T;X₁₀ is selected from the group consisting of G, R, and V;X₁₁ is selected from the group consisting of K and T;X₁₂ is selected from the group consisting of A, G, N, and T;X₁₄ is selected from the group consisting of I and T;any X not specified optionally has the amino acid sequence of thecorresponding position in SEQ ID NO:1; and the polypeptide optionallycomprising 1-4 additional amino acids;

(b) region 2 (amino acids 42-49 of SEQ ID NO:1) wherein the polypeptidecomprises the structure:

X₄₂-X₄₃-X₄₄-X-₄₅-X₄₆-X₄₇-X₄₈-X₄₉

whereinX₄₂ is selected from the group consisting of I, R, T, and V;X₄₄ is selected from the group consisting of I, L, and V;X₄₆ is selected from the group consisting of A, R, S, and Y;X₄₇ is selected from the group consisting of G, H, K, and R;X₄₈ is selected from the group consisting of K, M, and R;X₄₉ is selected from the group consisting of K, L, P, Q, R, and V; andany X not specified optionally has the amino acid sequence of thecorresponding position in SEQ ID NO:1; and

(c) region 3 (amino acids 62-78 of SEQ ID NO:1) wherein the polypeptidecomprises the structure:

X₆₂-X₆₃-X₆₄-X₆₅-X₆₆-X₆₇-X₆₈-X₆₉-X₇₀-X₇₁-X₇₂-X₇₃-X₇₄-X₇₅-X₇₆-X₇₇-X₇₈

whereinX₆₂ is selected from the group consisting of E, H, and Q;X₆₃ is selected from the group consisting of K and R;X₆₆ is selected from the group consisting of S and T;X₆₈ is selected from the group consisting of H, Q, and R;X₇₀ is selected from the group consisting of L and V;X₇₁ is selected from the group consisting of L, M, V, W, and Y;X₇₂ is selected from the group consisting of F, I, L, R, and V;X₇₃ is selected from the group consisting of F, L, and V;X₇₄ is selected from the group consisting of G, L, P, R, and S;X₇₅ is selected from F, G, K, R, and S;X₇₆ is selected from the group consisting of A, E, G, L, N, R, and V;X₇₇ is selected from the group consisting of E, G, R, and T, or isabsent;X₇₈ is selected from the group consisting of A, G, P, and S, or isabsent; andwherein X₃₄ is selected from D, E, and G, and any X not specified insaid Ubv polypeptide optionally has the amino acid sequence of thecorresponding position in SEQ ID NO:1; or a fragment thereof, whereinthe sequence of said Ubv polypeptide does not consist of SEQ ID NO:1.

As noted above, any X not specified can optionally have the amino acidsequence of the corresponding position in SEQ ID NO:1 or, alternatively,the sequence of the corresponding position in any of the specific Ubv'slisted herein, if different from that of SEQ ID NO:1.

In some embodiments, the Ubv polypeptide includes a substitution in oneor more position selected from the group consisting of X₈, X₁₁, and X₇₃of the amino acid sequence of SEQ ID NO:1. For example, the Ubvpolypeptide may include one or more substitution selected from L8G,K11T, and L73F. Furthermore, the Ubv polypeptide may further including asubstitution in position X₄₂ and/or X₆₈. (e.g., R42I in position X₄₂and/or X₆₈) and/or H68R. In another option, the Ubv polypeptide mayinclude one or more of the substitutions noted above (e.g., all of saidsubstitutions) and also an amino acid substitution in one or moreposition selected from the group consisting of X₉, X₁₀, X₁₂, X₄₆, X₄₇,X₄₉, X₆₂, X₆₃, X₇₂, X₇₆, X₇₇, and X₇₈ (e.g., one or more of T9A, G10R,T12A, A46S, G47R, Q49L, Q62H, K63R, R72I, R76N, G77E, and G78S). In aspecific example, the Ubv polypeptide includes each of the substitutions(Fw7.1). In other examples, the Ubv polypeptide includes one or moresubstitution selected from L8G, K11T, and L73F (e.g., all of said threesubstitutions) and one of the following sets of substitutions: (a) G10V,T12N, T14I, R42T, Q62E, T66S, H68R, R74G, R76L, 77T, and 78A (Fw7.2);(b) Q2H, F4L, G10V, R42I, I44V, A46Y, H68Q, V70L, R74L, 77R, and 78P(Fw7.3); or (c) T9A, T12N, R42I, A46S, Q49L, Q62H, K63R, H68R, R72I,R76N, 77E, and 78A (Fw7.4).

In another aspect, the invention provides Ubv polypeptide that includeone or more amino acid substitution selected from the group consistingof A12G, I42R or V, L49R, H62Q, R63K, and G75R in the amino acidsequence of Fw7.1, wherein amino acids 77 and 78 are optional. In oneexample, the Ubv polypeptide includes the following substitutions: A10G,I42V, L49R, H62Q, R63K, I72V, R74G, G75R, and G76R (Fw7.5), whereinamino acids 77 and 78 are optional. In other examples, the Ubvpolypeptide of claim 10, including the following substitutions: (a)A10G, L49R, H62Q, R63K, R74P, and G75S (Fw7.6); (b) A10G, I42R, S46A,R47G, K48R, L49R, H62Q, R63K, L71Y, I72V, and G75R (Fw7.7); (c) I42R,S46A, R47G, H62Q, R63K, L71W, I72F, F73L, G75F, and G76V (Fw7.8); (d)A10G, L49R, H62Q, R63K, L71M, I72V, R74S, and G75K (Fw7.9); (e) A9T,A10G, I42V, I44L, L49V, H62Q, R63K, L71W, I72L, F73L, and G75R (Fw7.10);(f) E36G, I42V, R47K, L49R, H62Q, R63K, and G75R (Fw7.11); (g) I42R,S46A, R47G, L49R, H62Q, R63K, I72L, G75R, and G76E (Fw7.12); (h) A9S,A10G, E36G, I44L, L49R, H62Q, R63K, R74S, and G76A (Fw7.13); (i) E36D,I42V, I44L, S46R, L49K, H62Q, R63K, V70L, L71W, F73V, and G75R (Fw7.14);(j) A10G, R47H, K48M, L49R, H62Q, R63K, and R74S (Fw.7.15); (k) A10G,E36G, I44L, L49P, H62Q, R63K, L71V, I72V, and G75R (Fw7.16); (l) A10G,E36G, I42R, S46A, R47G, L49R, H62Q, R63K, and T66S (Fw7.17); and (m)I42R, L49R, H62Q, R63K, I72L, and R74P (Fw7.18); wherein amino acids 77and 78 are optional.

In a third aspect, the invention provides Ubv polypeptides that includethe amino acid sequence of Fw11.1 (SEQ ID NO:20), optionally including1-10, e.g., 1-5, or 1-3 substitutions, which optionally may be in theregion of amino acids 1-25, 5-20, 10-19, or 11-17. For example, one ofthe following sets of substitutions may be included: (a) S12Y, S14T, andN17H (Fw11.2; SEQ ID NO:21); (b) S14T and Y15F (Fw11.3; SEQ ID NO:22);(c) S12Y (Fw11.4; SEQ ID NO:23); (d) S14T (Fw11.5; SEQ ID NO:24); (e)S14T, Y15F, and N17D (Fw11.6; SEQ ID NO:25); (f) S14T and N17H (Fw11.7;SEQ ID NO:26); (g) S12Y and S14T (Fw11.8; SEQ ID NO:27); (h) Y15F(Fw11.9; SEQ ID NO:28); (i) N17H (Fw11.10; SEQ ID NO:29); (j) S14N andY15F (Fw11.11; SEQ ID NO:30); (k) N17D (Fw11.12; SEQ ID NO:31); (l) S12Hand N17Y (Fw11.13; SEQ ID NO:32); (m) S12Y and S15F (Fw11.14; SEQ IDNO:33); (n) K11R and S14T (Fw11.15; SEQ ID NO:34); (o) S12T (Fw11.16;SEQ ID NO:35); and (p) S12A (Fw11.17; SEQ ID NO:36).

In a fourth aspect, the invention provides a Ubv polypeptide including asequence selected from the group consisting of SEQ ID NOs:2-36, or avariant thereof including a sequence that is at least 90% (e.g., 95%,97%, or 99%) identical to a sequence selected from the group consistingof SEQ ID NOs:2-36, or a fragment thereof. Optionally, the variantsequence (e.g., a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) occurs in the region of amino acids 1-25, 5-20, 10-19, or 11-17.

In a fifth aspect, the invention provides nucleic acid molecules (e.g.,isolated nucleic acid molecules) encoding a Ubv polypeptide as describedherein, as well as recombinant expression vectors including one or moreof such nucleic acid molecule, and host cells including one or more ofsaid nucleic acid molecules or recombinant expression vectors.

In a sixth aspect, the invention provides methods for obtainingubiquitin variant polypeptides that bind to a Skp1-F-box proteincomplex. These methods include randomizing sequences in region 1, region2, and/or region 3 of ubiquitin to create a library of variants, andscreening the library of variants for binding to the F-box protein ofthe Skp1-F-box protein complex, or a fragment thereof, optionallywherein region 1 includes up to 8 amino acids in addition to thosepresent in ubiquitin. In various examples, the F-box protein includesFbw7 or Fbw11, or a fragment thereof.

In a seventh aspect, the invention provides methods of modulating theactivity of an Skp1-Cul1-F-box (SCF) E3 ligase (e.g., and SCF E3 ligaseincluding Fbw7 or Fbw11) in a cell. These methods include contacting thecell with an agent that alters binding of Cul1 to a complex includingSkp1 and an F-box protein in the cell. The agent can optionally decreasethe activity of the SCFE3 ligase and/or decrease or inhibit binding ofCul1 to the complex including Skp1 and an F-box protein. Furthermore,the agent may optionally include a Ubv polypeptide, a nucleic acidmolecule encoding a Ubv polypeptide, or a fragment thereof (e.g., suchmolecules as described herein). In addition, the agent may havespecificity for a particular SCF E3 ligase, or may be active againstmore than one SCF E3 ligase. In various examples, the cell is a cancercell, which optionally is within a subject having cancer. Thus, theinvention includes methods of treating cancer in a subject, in which theactivity of an Skp1-Cul1-F-box (SCF) E3 ligase in a cell of the subjectis modulated as described herein.

In an eighth aspect, the invention provides methods of identifying anagent that modulates the activity of an SCF E3 ligase in a cell. Thesemethods include contacting a cell expressing an SCF E3 ligase with acandidate agent (e.g., a small molecule compound), and determiningwhether the agent affects the binding of Cul1 to a complex includingSkp1 and an F-box protein (e.g., by use of an immunoprecipitationassay). In various examples, the cell further expresses a Ubvpolypeptide, such as a Ubv polypeptide as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Ubvs selected for binding to the Skp1tr-Fbw7 complex. (A)Schematic of SCF E3 ligase. (B) Sequence alignment of selected Ubvs.Library 1 sequence is shown, where residue letters indicate the wt Ubsequence that was soft randomized and X denotes positions that werecompletely randomized. Only diversified positions are shown and residuesin Ubvs conserved as wt Ub are indicated by dashes. Sequences showingconservation across selected Ubvs are highlighted in grey. (C) Bindingof selected Ubvs to Skp1tr in complex with F-box-WD40^(Fbw7),F-box^(Fbw7), or F-box^(Fbw11). Ubv-phage binding was measured by ELISAwith the indicated immobilized proteins. (D and E) Binding of purifiedUbv.Fw7.1 or Ubv.Fw7.2 to Skp1tr-F-box^(Fbw7) as measured by ELISA. Datafrom a typical experiment are shown and the binding values arerepresented as mean±S.E. of at least two experiments. (D) IC₅₀ valueswere calculated by competitive ELISA as the concentration ofSkp1tr-F-box^(Fbw7) in solution that blocked 50% of Ubv binding toimmobilized Skp1tr-F-box^(Fbw7). (E) EC₅₀ values were calculated bydirect-binding ELISA as the concentration of Ubv at which 50% of thesaturation signal is achieved for binding to immobilized Skp1tr-F-box^(Fbw7) complex.

FIG. 2. Structural and mutational analysis of the interactions betweenUbv.Fw7.1 and the Skp1tr-F-box^(Fbw7) complex. (A) Structure ofUbv.Fw7.1 in complex with Skp1tr-F-box^(Fbw7). Ubv regions (Regions 1-3)that were diversified in Library 1 are labeled. (B) Details of themolecular interactions between Ubv.Fw7.1 and Skp1tr-F-box^(Fbw7) showingresidues that are mutated relative to wt Ub and are critical forbinding. Skp1tr and Fbw7 residues are denoted by “S” and “F”superscripts, respectively. (C) Affinities of Ubv.Fw7.1 back-mutants forSkp1tr-F-box^(Fbw7). Ubv.Fw7.1^(Min) lacks Ub tail (residues 75-78) andcontains only six mutations relative to wt Ub (L8G, G1 OR, K11T, R42I,H68R, and L73F). “NB” indicates no detectable binding. (D) Superpositionof Skp1tr-F-box^(Fbw7)-Ubv.Fw7.1 complex with Skp1tr-F-box^(Fbl1)-Cul1complex (PDB: 1 LDK). (E) Comparison of the Ubv.Fw7.1-binding andpredicted Cul1-binding surfaces on Skp1tr-F-box^(Fbw7).Skp1tr-F-box^(Fbw7) residues interacting with Ubv.Fw7.1 or predicted tointeract with Cul1 (by comparison to the Skp1-F-box^(Fbl1)-Cul1 complex)are shown as stick and shaded according to predicted interactions:medium grey interacts with Cul1 and Ubv.Fw7.1; light grey, interactswith Cul1 only; dark grey, interacts with Ubv.Fw7.1 only. Residues thatwere subjected to mutagenesis are labeled. (F) Effects of substitutionsin Skp1tr or the F-box^(Fbw7) domain on the binding of Skp1tr-F-box^(Fbw7) to Ubv.Fw7.1 or Cul1N-terminal domain (NTD).

FIG. 3. Ubvs selected for binding to the F-box^(Fbw7) or F-box^(Fbw11)domain in complex with full-length Skp1. (A) Ubvs selected from Library2 for binding to Skp1-F-box^(Fbw7). Positions that were soft randomizedin the library are shown and residues conserved as Ubv.Fw7.1 sequenceare indicated by dashes. Positions that diverge from the Ubv.Fw7.1sequence but show consensus amongst the selected sequences are boxed andconserved residues at these positions are shaded grey. (B) Affinities ofUbv.Fw7.5, Ubv.Fw11.1, and Ubv.Fw11.2 for different Skp1-F-boxcomplexes. “NB” indicates no detectable binding and “WB” indicates weakbinding for which IC₅₀ values were >5000 nM. Sequence of Ubv bindingregion (F-box residues located within 10 Å of Ubv in the structure ofSkp1tr-F-box^(Fbw7-)Ubv.Fw7.1 complex) is shown for each F-box protein.Conserved positions are shaded grey and Fbw7 residues important forbinding to Ubv.Fw7.1 (FIG. 2F) are boxed. (C) The sequences andaffinities of Ubv.Fw11.1 and its derivatives selected for binding toSkp1-F-box^(Fbw11). Only the sequence in Region 1 that differs fromUbv.Fw7.5 is shown, and residues conserved as Ubv.Fw11.1 sequence areindicated by dashes.

FIG. 4. Biological activity of Ubvs in HEK293T cells. (A) Ubvinteraction partners identified by mass spectrometry of FLAG-Ubvimmunoprecipitates from cell lysates. Spectral count refers to thenumber of peptides corresponding to each identified protein. Onlyproteins relevant to SCF ligases are shown (see Table 4 for completelist of detected proteins). (B) Expression of Fw7.5 Ubv in monomer ordimer disrupts interaction of Fbw7 with Cul1. HA-Fbw7 immunoprecipitateswere probed for FLAG-Cul1 and endogenous Skp1, in the absence orpresence of FLAG-Ubv expression. (C) Expression of Ubv.Fw11.2 Ubv indimer format, but not monomer format, disrupts interaction between Fbw11and Cul1. Analysis performed as described in (B). (D) and (E) Expressionof Ubv.Fw7.5 (D) and Ubv.Fw11.2 (E) in monomer or dimer formatstabilizes the SCF^(Fbw7) (Cyclin E and c-Myc) and SCF^(Fbw11) (Cdc25Aand Wee1) substrates respectively. Cells were transiently transfectedwith either siRNA molecules (positive control), empty vector (Vector),or vectors expressing FLAG-Ubv. Cells were treated with Cycloheximide(CHX) for the indicated time points and cell lysates were probed withantibodies against the indicated proteins. Quantification of relativesubstrate levels was performed using ImageJ and represents average oftwo independent experiments (FIG. 4D, 8C for c-Myc and Cyclin E and FIG.4E, 8D for Cdc25A and Wee1). (E) The effect of Fbw11 siRNA treatment andUbv.Fw11.2 expression was assessed in the background of Fbw1 siRNAtreatment.

FIG. 5. Ubv libraries. (A) Regions 1, 2 and 3 targeted in the librarydesigns are shown. Only positions relevant to the library design areincluded and residues that differ from wt Ub sequence are highlighted ingrey. Positions subjected to soft-randomization are boxed and positionssubjected to complete randomization are indicated by X. (B)Skp1tr-F-box^(Fbw7)-Ubv.Fw7.1 complex showing Ub positions diversifiedin the libraries. Regions 1, 2, and 3 are shown in separate panels andtargeted positions are shown as grey spheres and numbered. Skp1tr andF-box^(Fbw7) are shaded light grey and Ubv is shaded dark grey. Loop1deleted in Skp1tr is indicated.

FIG. 6. Additional in vitro experiments (A) Alignment of all Skp1constructs tested in this study. Loop 1 (residues 38-43) and Loop 2(residues 70-81) indicate residues deleted in truncated version of Skp1(Skp1tr). (B) and (C) Binding of Cul1N-terminal domain (NTD) toSkp1-F-box^(Fbw7) and Skp1tr-F-box^(Fbw7) complexes demonstratesimportance of Skp1 Loop 1 and Loop 2 residues for interaction with Cul1.Binding was measured by Surface Plasmon Resonance (SPR) analysis andobtained traces are shown for Cul1 NTD interaction withSkp1-F-box^(Fbw7) (B) and Skp1tr-F-box^(Fbw7) (C). Skp1-F-box complexeswere immobilized and Cul1 NTD was injected at different concentrations(100, 33, 11, 3.7 and 1.2 nM). Black lines represent fits to a simple1:1 Langmuir binding isotherm model. In the case of binding toSkp1-F-box^(Fbw7) only 3 lowest Cul1 concentrations (11 nM Cul1, 3.7 nMCul1, and 1.2 nM Cul1) were used to fit the data, since higherconcentrations exceeded estimated affinity of the interaction>1000 fold.Estimated k_(on), k_(on) rates and Kd of the interactions are indicated.(D) Structure of Skp1tr-F-box^(Fbl1)-Cul1 (PDB: 1LDK) complexhighlighting positions of loops deleted in Skp1tr in relation to Cul1binding surface. Location of loops deleted in Skp1tr are indicated. (E)Ubv.Fw7.1 promotes dissociation of Cul1 from the SCF^(Fbw7) complex.SCF^(Fbw7) complex containing GST-tagged Skp1tr on glutathioninesepharose resin was incubated for 1 hour with increasing amounts ofUbv.Fw7.1. Cul1 remaining bound to the resin was detected by westernblot. (F) Ubv.Fw7.1 blocks ubiquitination activity of SCF^(Fbw7). Afunctional SCF^(Fbw7) complex was assembled from theSkp1tr-(F-box-WD40)^(Fbw7) and Cul1-Rbx1 complexes purified separately.Sic1, which is a natural substrate of yeast Fbw7 but is also recognizedby human Fbw7, was used as the substrate. The ubiquitination reactioncontaining E1 (0.5 μM), E2 (14 μM), and SCF^(Fbw7) (0.4 μM) wasinitiated by adding Ub (25 μM) to purified components in the absence andpresence of Ubv.Fw7. 1 (25 μM). The products of Sic1 (0.4 μM)ubiquitination were visualized by western blotting at the indicated timepoints. (G) Skp1 Loop 1 (residues 38-43) interferes with binding toUbv.Fw7.1, while Skp1 Loop 2 (residues 70-81) has no effect on binding.Binding was tested by protein ELISA and dose response curves ofUbv.Fw7.1 binding to different Skp1 constructs in complex withF-box^(Fbw7) domain are shown. (H) Structure ofSkp1tr-F-box^(Fbw7)-Ubv.Fw7.1 complex highlighting positions of loopsdeleted in Skp1tr. Positions that showed different preferences in Ubvselected against Skp1-F-box^(Fbw7) versus parental Ubv.Fw7.1 sequenceare shown as grey spheres. Position 75 was not defined in the structureand its projected location is shown.

FIG. 7. Ubvs selected for binding to the Skp1-F-box^(Fbw11) complex.Only positions in Region 1 that were diversified in the library areshown and residues conserved as Ubv.Fw11.1 sequence are indicated bydashes.

FIG. 8. Additional intracellular experiments. (A) Interaction betweenFLAG-Ubv.Fw7.5 and exogenously expressed HA-Fbw7 or HA-Fbl1 in celllysates. FLAG immunoprecipitates were separated on gel electrophoresisand probed for the presence of the indicated HA-tagged F-box proteins.Ubv.Fw7.5 co-immunoprecipitates significant levels of Fbw7, whereas Fbl1presence in immunoprecipitates could not be detected by this assay. Itshould be noted that the levels of Fbw7-HA in cell lysates aresignificantly greater in the presence of Ubv.Fw7.5 expression; this isthe result of inhibition of Fbw7 auto-ubiquitination (Welcker, 2013) bythe Ubv.Fw7.5. (B) Validation of Fbw7, Fbw1, and Fbw11 siRNA. Cellsexpressing FLAG-Fbw7 (top panel) and FLAG-Fbw1, FLAG-Fbw11 andFLAG-Fbw1+HA-Fbw11 (bottom panel) were treated either with control siRNA(C) or the siRNAs directed against the indicated proteins. (C) and (D).Expression of Ubv.Fw7.5 (C) and Ubv.Fw11.2 (D) dimer or monomerselectively stabilizes substrates of SCF^(Fbw7) and SCF^(Fbw11)respectively, but not other SCF ligases. Cells were transientlytransfected with either siRNA molecules (positive control), empty vector(vector), or vectors expressing FLAG-Ubv. Cells were treated withCycloheximide (CHX) for the indicated time points and cell lysates wereprobed with endogeneous antibodies against the indicated proteins(substrate (F-box protein)). (E) Expression of Ubv.Fw11.2 does notaffect stability of Cdc25A and Wee1 in the background of Fbw1 siRNAtreatment. (F) and (G) Distribution of G1-phase, G2-phase, and S-phasepopulations in cells expressing Ubv.Fw7.5 (F) and Ubv.Fw11.2 (G). Cellswere transiently transfected with either siRNA molecules (positivecontrol), empty vector (vector), or vectors expressing FLAG-Ubv.Analysis of cell cycle kinetics was determined by Hoechst dye (nucleicacid stain) staining, followed by flow cytometry analysis. The graphcombines data from three biological replicates and mean±S.E. is shown.(G) The effect of Fbw11 siRNA treatment and Ubv.Fw11.2 expression wasassessed in the background of Fbw1 siRNA treatment.

DETAILED DESCRIPTION

The attachment of ubiquitin (Ub) to target proteins involves theactivities of Ub-activating enzymes (E1 enzymes), Ub-conjugating enzymes(E2 enzymes), and Ub ligases (E3 enzymes). Ubiquitination can alter theproperties of target proteins in many ways, including directing them tothe proteasome for degradation, as well as altering their cellularlocalization, activities, and/or interactive properties with respect toother proteins. Modification of ubiquitination thus provides anopportunity to modify a very wide variety of different cellularfunctions, in many contexts.

The present invention provides ubiquitin (Ub) variants, or UbVs, whichtarget a particular family of E3 ligases, SCF E3 ligases. The inventionalso provides nucleic acid molecules encoding such UbVs, as well asrelated vectors and cells. In addition, the invention provides methodsfor identifying and characterizing new SCF E3 ligase-specific UbVs.Furthermore, the invention provides methods of using UbV polypeptidesand related molecules. Examples of the latter include, for example,methods of identifying other modulators of SCF E3 ligase activity, aswell as therapeutic methods involving SCF E3 ligase activity modulation.These and other aspects of the invention are described further, asfollows.

The UbVs of the invention bind to or otherwise impact the activity ofone or more SCF E3 ligase. The UbVs of the invention can have broadactivity, against a wide range of SCF E3 ligases or, alternatively, maybe relatively specific, modulating the activity of a small, relatedsubset of SCF E3 ligases or even only a single, specific SCF E3 ligase.The UbVs modulate the activity of an SCF E3 ligase by, for example,blocking or decreasing the ligase activity. The modulation (blocking ordecreasing of activity) can be by direct interaction with an SCF E3ligase. In one example of such an interaction, a UbV binds to an SCF E3ligase with greater affinity than Ub, resulting in competitiveinhibition. In one specific example, the UbV binds to the interface ofSkp1 and F-box proteins and thereby prevents Cul1 binding to the samesurface and inhibits SCF E3 ligase activity by disrupting complexformation.

SCF E3 ligases that can be targeted by the UbVs of the invention includethose containing F-box proteins selected from the Fbw, Fbl, and Fbofamilies. For example, the SCF E3 ligases can include human Fbw7 orFbw11 F-box proteins. Additional examples include: Fbw1, Fbw2, Fbw4,Fbw5, Fbw8, Fbw9, Fbw10, Fbw12, Fbl1, Fbl2, Fbl3, Fbl4, Fbl5, Fbl6,Fbl7, Fbl8, Fbl10, Fbl11, Fbl12, Fbl13, Fbl14, Fbl15, Fbl16, Fbl17,Fbl19, Fbl20, Fbl21, Fbl22, Fbo1, Fbo2, Fbo3, Fbo4, Fbo5, Fbo6, Fbo7,Fbo8, Fbo9, Fbo10, Fbo11, Fbo15, Fbo16, Fbo17, Fbo18, Fbo20, Fbo21,Fbo22, Fbo24, Fbo25, Fbo27, Fbo28, Fbo30, Fbo31, Fbo32, Fbo33, Fbo34,Fbo36, Fbo38, Fbo39, Fbo40, Fbo41, Fbo42, Fbo43, Fbo44, Fbo45, Fbo46,Fbo48. Additional SCF E3 ligases that can be targeted include SCF E3ligases from other eukaryotic species and eukaryotic-like F-boxeffectors from bacteria.

The UbVs of the invention comprise one or more mutation (e.g.,substitution, deletion, addition, or modification) within any region orregions of a wild-type Ub. Using the sequence of human ubiquitin as areference (SEQ ID NO:1), the UbVs can have mutations (e.g.,substitutions, deletions, or insertions) in one or more (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) amino acid in one or more ofregion 1 (amino acids 2-14), region 2 (42-49), or region 3 (62-78). Awild-type variant having two C-terminal glycines added to the sequenceof SEQ ID NO:1 can also serve as a basis for generating UbVs.Furthermore, in addition to human Ub, the invention features UbVsobtained on the basis of Ub from other species and sources.

The sequence of Ub and specific examples of UbVs of the invention areprovided in Table 1.

TABLE 1 SEQ ID NO Target Ubv Ubv sequence  1 UbMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG[GG]  2 Skp1 (38-43Δ, 70-77Δ, Fw7.1MQIFVKTGARTAITLEVEPSDTIENVKAKIQDKEGIPPDQQ K78G E79S K80GILIFSRKLLEDGRTLSDYNIHRESTLRLVLIFRGNES R81G)-Fbw7  3Skp1 (38-43Δ, 70-77Δ, Fw7.2 MQIFVKTGTVTNIILEVEPSDTIENVKAKIQDKEGIPPDQQK78G E79S K80G TLIFAGKQLEDGRTLSDYNIEKESSLRLVLRFGGLTA R81G)-Fbw7  4Skp1 (38-43Δ, 70-77Δ, Fw7.3 MHILVKTGTVTTITLEVEPSDTIENVKAKIQDKEGIPPDQQK78G E79S K80G ILVFYGKQLEDGRTLSDYNIQKESTLQLLLRFLGGRP R81G)-Fbw7  5Skp1 (38-43Δ, 70-77Δ, Fw7.4 MQIFVKTGAGTNITLEVEPSDTIENVKAKIQDKEGIPPDQQK78G E79S K80G ILIFSGKLLEDGRTLSDYNIHRESTLRLVLIFRGNEA R81G)-Fbw7  6SKp1-Fbw7 Fw7.5 MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFGRR  7 SKp1-Fbw7 Fw7.6MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKEGIPPDQQILIFSRKRLEDGRTLSDYNIQKESTLRLVLIFPSG  8 SKp1-Fbw7 Fw7.7MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGRRLEDGRTLSDYNIQKESTLRLVYVFRRG  9 SKp1-Fbw7 Fw7.8MQIFVKTGARTAITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKLLEDGRTLSDYNIQKESTLRLVWFLRFV 10 SKp1-Fbw7 Fw7.9MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKEGIPPDQQILIFSRKRLEDGRTLSDYNIQKESTLRLVMVFSKG 11 SKp1-Fbw7 Fw7.10MQIFVKTGTRTGITLEVEPSDTIENVKAKIQDKEGIPPDQQVLLFSRKVLEDGRTLSDYNIQKESTLRLVWLLRRG 12 SKp1-Fbw7 Fw7.11MQIFVKTGARTAITLEVEPSDTIENVKAKIQDKGGIPPDQQVLIFSKKRLEDGRTLSDYNIQKESTLRLVLIFRRG 13 SKp1-Fbw7 Fw7.12MQIFVKTGARTAITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKRLEDGRTLSDYNIQKESTLRLVLLFRRE 14 SKp1-Fbw7 Fw7.13MQIFVKTGSRTGITLEVEPSDTIENVKAKIQDKGGIPPDQQILLFSRKRLEDGRTLSDYNIQKESTLRLVLIFSGA 15 SKp1-Fbw7 Fw7.14MQIFVKTGARTAITLEVEPSDTIENVKAKIQDKDGIPPDQQVLLFRRKKLEDGRTLSDYNIQKESTLRLLWIVRRG 16 SKp1-Fbw7 Fw7.15MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKEGIPPDQQILIFSHMRLEDGRTLSDYNIQKESTLRLVLIFSGG 17 SKp1-Fbw7 Fw7.16MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKGGIPPDQQILLFSRKPLEDGRTLSDYNIQKESTLRLVVVFRRG 18 SKp1-Fbw7 Fw7.17MQIFVKTGARTGITLEVEPSDTIENVKAKIQDKGGIPPDQQRLIFAGKRLEDGRTLSDYNIQKESSLRLVLIFRGG 19 SKp1-Fbw7 Fw7.18MQIFVKTGARTAITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFSRKRLEDGRTLSDYNIQKESTLRLVLLFPGG 20 Skp1-Fbw11 Fw11.1MQIFVKTYPYKSGSYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 21 Skp1-Fbw11 Fw11.2MQIFVKTYPYKYGTYHHNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 22 Skp1-Fbw11 Fw11.3MQIFVKTYPYKSGTFHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 23 Skp1-Fbw11 Fw11.4MQIFVKTYPYKYGSYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 24 Skp1-Fbw11 Fw11.5MQIFVKTYPYKSGTYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 25 Skp1-Fbw11 Fw11.6MQIFVKTYPYKSGTFHDNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 26 Skp1-Fbw11 Fw11.7MQIFVKTYPYKSGTYHHNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 27 Skp1-Fbw11 Fw11.8MQIFVKTYPYKYGTYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 28 Skp1-Fbw11 Fw11.9MQIFVKTYPYKSGSFHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 29 Skp1-Fbw11 Fw11.10MQIFVKTYPYKSGSYHHNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 30 Skp1-Fbw11 Fw11.11MQIFVKTYPYKSGNFHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 31 Skp1-Fbw11 Fw11.12MQIFVKTYPYKSGSYHDNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 32 Skp1-Fbw11 Fw11.13MQIFVKTYPYKHGSYHYNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 33 Skp1-Fbw11 Fw11.14MQIFVKTYPYKYGSFHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 34 Skp1-Fbw11 Fw11.15MQIFVKTYPYRSGTYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 35 Skp1-Fbw11 Fw11.16MQIFVKTYPYKTGSYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR 36 Skp1-Fbw11 Fw11.17MQIFVKTYPYKAGSYHNNYTITLEVEPSDTIENVKAKIQDKEGIPPDQQVLIFSRKRLEDGRTLSDYNIQKESTLRLVLVFG RR

In addition to UbVs having the sequences set forth above, the inventionincludes variants of these and other UbVs. Thus, for example, theinvention includes polypeptides having at least 80%, 85%, 95%, or 99%sequence identity to a UbV, such as a UbV described herein. Theinvention also includes UbV variants having one or more (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, or more) substitutions (e.g., conservative amino acidsubstitutions) and/or deletions relative to a sequence provided herein.

A “conservative” amino acid substitution as used herein, is one in whichone amino acid residue is replaced with another amino acid residuehaving similar properties. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid, glutamic acid, asparagine, andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine. In more detail, conserved amino acid substitutions involvereplacing one or more amino acids of the polypeptides of the inventionwith one or more amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade, the resulting molecule may be functionally equivalent or similarto the original molecule. Changes that result in production of achemically equivalent or chemically similar amino acid sequence areincluded within the scope of the invention. In various examples, ahydrophobic residue, such as glycine, can be substituted for anotherhydrophobic residue such as alanine. An alanine residue may besubstituted with a more hydrophobic residue such as leucine, valine, orisoleucine. A negatively charged amino acid, such as aspartic acid, maybe substituted for glutamic acid. A positively charged amino acid suchas lysine may be substituted for another positively charged amino acid,such as arginine. The phrase “conservative substitution” also includesthe use of a chemically derivatized residue in place of anon-derivatized residue, provided that such polypeptide displays arequisite activity.

The invention includes polypeptides that comprise the sequences of theUbVs described herein, in addition to other sequences. Thus, forexample, the invention includes fusion proteins comprising the UbVs (andvariants thereof) described herein (e.g., fusions with GST, His, Flag,or Myc tags). In addition, the invention includes fragments of the UbVs(and variants thereof) described herein. Such fragments include, forexample, a UbV (or variant thereof) having 1-30 (e.g., 2-25, 4-30, or5-10) amino acids deleted from either or both ends of the UbV (orvariant thereof). Internal deletions are also included in the invention.The fragments can optionally be comprised within a fusion protein, asdescribed above in connection with full-length UbVs. Optionally, UbVvariants and fragments maintain, at least in part, one or moreactivities of the UbV from which they are derived. The fragments canfurther optionally comprise one or more region of a UbV, as describedherein (e.g., region 1, region 2, region 3, region 1 and 2, region 2 and3, etc.) The UbVs of the invention can be used to obtain or designpeptide mimetics, which are also included in the invention. Peptidemimetics include synthetic structures that may serve as substitutes forpeptides in interactions between molecules, and include syntheticstructures which can optionally contain amino acids and/or peptidebonds, but are designed to retain the desired structural and functionalfeatures and thus may be suitable substitutes of the peptide inhibitoranalog disclosed herein. Peptide mimetics also include moleculesincorporating peptides into larger molecules with other functionalelements (e.g., as described in WO 99/25044). Peptide mimetics alsoinclude peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad.Sci. USA 89:9367), and peptide libraries containing peptides of adesigned length representing all possible sequences of amino acidscorresponding to an isolated peptide of the disclosure. Peptide mimeticsmay be designed based on information obtained by systematic replacementof L-amino acids by D-amino acids, replacement of side chains withgroups having different electronic properties, and by systematicreplacement of peptide bonds with amide bond replacements. Localconformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

The UbVs described herein can be made using standard methods including,for example, recombinant methods. The UbVs may also be prepared bychemical synthesis using techniques well known in the art such as solidphase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964);Frische et al., J. Pept. Sci. 2(4): 212-22 (1996)) or synthesis inhomogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E.Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)). The UbVs of theinvention typically comprise naturally occurring amino acids. However,UbVs including one or more non-naturally occurring amino acid are alsoincluded in the invention.

In addition to the UbVs described above, the invention provides nucleicacid molecules encoding the UbVs (e.g., nucleic acid molecules encodingUbVs of any one of SEQ ID NOs:2-36) and variants thereof, as describedherein.

The term “nucleic acid molecule” as used herein refers to a sequence ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted sequences comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Thenucleic acid molecules of the present invention can be ribonucleic (RNA)or deoxyribonucleic acids (DNA), and can contain naturally occurringbases including adenine, guanine, cytosine, thymidine, and uracil. Thesequences can also contain modified bases such as xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine andother 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil, and5-trifluoro cytosine.

The term “isolated and purified” as used herein refers to a nucleic acidmolecule, polypeptide, or peptide that is substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors, or other chemicals when chemically synthesized.An “isolated and purified” nucleic acid molecule is also substantiallyfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) from whichthe nucleic acid molecule is derived.

Nucleic acid molecules encoding the UbVs can optionally be comprisedwithin a vector, such as an expression vector. Exemplary vector typesinclude cosmids, plasmids, or modified viruses (e.g. replicationdefective retroviruses, adenoviruses, and adeno-associated viruses). Theexpression vectors can include a nucleic acid molecule encoding a UbV,as well as operatively linked regulatory sequences that are selectedbased on the type of host cells in which expression is to occur.“Operatively linked” is intended to mean that the nucleic acid moleculeis linked to regulatory sequences in a manner that allows expression ofthe nucleic acid under the control of the regulatory element.

The invention thus includes recombinant expression vectors comprising anucleic acid molecule encoding a UbV, as described herein, andoptionally regulatory sequences that direct transcription of the nucleicacid molecule. Suitable regulatory sequences are known in the art andcan be obtained from a variety of sources, including bacterial, fungal,viral, mammalian, and insect genes. Selection of appropriate regulatorysequences is dependent on the host cell chosen, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector. Furthermore, the recombinant expressionvectors may also contain a selectable marker gene which facilitates theselection of host cells transformed or transfected with a recombinantmolecule of the disclosure. Examples of selectable marker genes aregenes encoding a protein such as G418 and hygromycin, which conferresistance to certain drugs, β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, or an immunoglobulin or portionthereof such as the Fc portion of an immunoglobulin optionally IgG.Transcription of the selectable marker gene is monitored by changes inthe concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors. It will be appreciated that selectable markers can beintroduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors can also contain genes that encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce transformed host cells, which are also included in theinvention. Suitable host cells include a wide variety of eukaryotic hostcells and prokaryotic cells. For example, the UbVs of the invention canbe expressed in mammalian, insect, yeast, or bacterial cells (e.g., E.coli).

The nucleic acid molecules of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing nucleic acid molecules are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (see, e.g., Itakura et al., U.S.Pat. No. 4,598,049; Caruthers et al., U.S. Pat. No. 4,458,066; andItakura, U.S. Pat. Nos. 4,401,796 and 4,373,071).

As noted above, the invention provides methods of identifying andcharacterized SCF E3 ligase-specific UbVs. Such UbVs can be obtained byscreening libraries of Ub variants, which can be generated byrandomizing the entire sequence of ubquitin (SEQ ID NO:1) or particularregions (e.g., one or more of regions 1 (2-14), 2 (42-49), and 3(62-78), which optionally may be truncated or expanded by, e.g., 1-10 or2-5 amino acids). Randomization can be achieved using standard methodsof genetic engineering. For example, variants can be created in which aparticular residue is replaced with a different amino acid, such that alibrary of variants comprising all 20 amino acids in each position(e.g., within one or more of regions 1, 2, and 3) is produced. In oneexample, randomization is performed to yield 75% wild type amino acidresidues and 25% mutated amino acid residues within, e.g., one or moreof regions 1, 2, and 3.

UbV libraries (e.g., phage display libraries) can be screened againstone or more SCF E3 ligase (e.g., see the list set forth above) and/or aportion of one or more SCF E3 ligase. In one example, the libraries arescreened against a complex including an F-box protein and Skp1, orfragments thereof. In one example, the F-box component of the complexincludes only an F-box domain and a substrate binding domain (e.g., aWD40 or LRR domain), while the Skp1 protein is full length or optionallyincludes truncations in one or more loops (e.g., loop 1 and/or loop 2;see below for example of Skp1tr). UbVs identified as binding to an SCFE3 ligase (or portion or fragment thereof) can then be subject tofurther characterization including, for example, assessment of bindingaffinity by EC₅₀ determination, specificity for particular SCF E3ligases (or subgroups thereof), structural features (e.g., byco-crystallization analysis), and effects on ubiquitination. The lattereffects of UbVs can be assessed using in vitro ubiquitination assays, aswell as in cell-based assays that assess the effects of a UbV ondownstream effects of ubiquitination involving a particular SCF E3ligase. Details of exemplary assays that can be used in this aspect ofthe invention are provided in the Examples, below.

In addition to being identified and characterized in various assays, asdescribed above, UbVs identified in the screening of libraries can besubject to further mutagenesis, in order to identify additional UbVshaving desirable features. Thus, for example, UbVs found to have adesirable property (e.g., binding specificity), but lacking anotherfeatures (e.g., binding affinity) can be further mutagenized andre-screened, optionally with the sequences of residues surmised bysequence analysis to be important with respect to the already obtaineddesirable property (e.g., binding specificity) maintained.

The invention also provides methods for modulating SCF E3 ligaseactivity. These methods include in vivo modulation of SCF E3 ligaseactivity by administration of a UbV as described herein, or a nucleicacid molecule encoding such a UbV (e.g., a nucleic acid molecule in anexpression or delivery vector, such as a vector as described herein) toa subject (e.g., a human patient). Ex vivo methods, in which a UbVpolypeptide or nucleic acid molecule is contacted with a cell or tissuethat is then introduced into a subject for therapeutic purposes, arealso included in the invention.

The therapeutic methods of the invention can be used in the preventionor treatment of diseases and conditions including, for example, cancer.Examples of cancer types that can be treated according to the methods ofthe invention include brain cancer, ovarian cancer, leukemia, lungcancer, colon cancer, CNS cancer, melanoma, myeloma, renal cancer,prostate cancer, pancreatic cancer, and breast cancer. Additionaldiseases or conditions that can be treated include sleep and metabolicdisorders, immune disorders, Hepatitis C virus-related conditions, andmuscle atrophy.

The invention also includes methods of identifying additional agentsthat can be used to modulate SCF E3 ligase activity, and thus which canbe used in the therapeutic methods described above. In such methods, acomplex comprising a UbV and an SCF E3 ligase (or a fragment or portionthereof, such as a complex of an F-box protein and Skp1 (or portionsthereof; see, e.g., above) is contacted with a candidate agent todetermine whether the candidate agent impacts the ability of the UbV tobind to the SCF E3 ligase (or fragment thereof). An agent that affectsthe binding (e.g., decreases or increases the binding) can be consideredas a candidate for modulation of SCF E3 ligase activity and, thus, maybe considered for use in a therapeutic method (e.g., see above). Suchcandidate agents can be tested in an in vitro ubiquitination assay or incell-based assays, such as those described herein. Candidate agents thatcan be screened in such assays include, e.g., peptides, nucleic acidmolecules, natural products, and small organic or inorganic molecules.Such agents may be present in the context of a library, which can betested in a high throughput manner.

The following non-limiting examples are illustrative of the presentdisclosure:

Experimental Examples

Selection of Ubv Binders for the Skp1tr-Fbw7 Complex

To investigate the potential of using Ubvs to target F-box familymembers, we used a naïve phage-displayed Ubv library (Ernst) (FIGS. 1 Band 5) to perform binding selections against Fbw7 in complex with Skp1.To facilitate structural characterization, we used Fbw7 and Skp1constructs that were previously used for structural studies but stillcontained all necessary functional elements required for E3 ligaseactivity. This included Fbw7 composed of F-box and WD40 domains(F-box-WD40^(Fbw7)) (Hao, 2007) and Skp1 with truncations in two loops(Skp1tr) (Schulman, 2000) (Table 2).

The selections yielded four unique binding Ubvs that shared commonmutations at several positions (FIG. 1 B), suggesting that they alllikely bind to a common site on the Skp1tr-Fbw7 complex. To determinethe region targeted by the selected Ubvs, we performed phageenzyme-linked immunosorbent assays (ELISAs) against Skp1tr complexedwith F-box-WD40^(Fbw7), Fbw7 F-box domain (F-box^(Fbw7)) or Fbw11 F-boxdomain (F-box^(Fbw11)). Surprisingly, the Ubvs did not target the WD40domain, which is known to interact with Ub (Pashkova, 2010) and smallmolecule inhibitors (Orlicky), but rather, specifically targetedF-box^(Fbw7) in complex with Skp1tr (FIG. 1 C). Relative affinities ofUbv.Fw7.1 and Ubv.Fw7.2 were measured for purified proteins with ELISAsthat determined half-maximum effective concentration of Ubv binding toimmobilized Skp1tr-F-box^(Fbw7) (EC₅₀) and half-maximum inhibitoryconcentration of Skp1tr-F-box^(Fbw7) in solution that inhibited bindingof Ubv to immobilized Skp1tr-F-box^(Fbw7) (IC₅₀). Since the IC₅₀ valuereflects the interaction between the two proteins in solution, itprovides a good estimate of the affinity (Lee, 2004). Ubv.Fw7.1exhibited the highest binding activity in both assay formats (IC₅₀=70 nMand EC₅₀=0.9 nM) and was chosen for further characterization (FIGS. 1 Dand E).

Structure of Ubv.Fw7.1 in Complex with Skp1tr-F-Box^(Fbw7)

We crystallized Ubv.Fw7.1 in complex with Skp1tr-F-box^(Fbw7) and solvedthe structure at 2.5 Å resolution by molecular replacement (FIGS. 2 Aand B and see Table 3 for X-ray data collection and refinementstatistics). Ubv.Fw7.1 makes extensive contacts with Skp1tr but alsomakes significant contacts with F-box^(Fbw7) (719 or 144 Å² of Ubvaccessible surface area buried, respectively). The structure ofSkp1tr-F-box^(Fbw7) in the ternary complex aligns closely with thepreviously determined structure of the Skp1tr-(F-box-WD40)^(Fbw7)complex (Hao, 2007), suggesting that Ubv.Fw7.1 does not induce majorconformational changes upon binding (RMSD of Skp1=0.93 Å and RMSD ofFbw7 (residues 279-313)=1.07 Å).

Although Ubv.Fw7.1 contains 15 substitutions relative to wild-type (wt)Ub and two additional C-terminal residues, back mutation analysisrevealed that only six substitutions (L8G, G1 OR, K11T, R42I, H68R andL73F) are responsible for most of the enhancement in binding toSkp1tr-Fbw7. A variant containing these six substitutions(Ubv.Fw7.1^(Min)) bound to Skp1tr-F-box^(Fbw7) only ˜20-fold weaker thanUbv.Fw7.1, but further back mutation of any of the six substitutionsgreatly reduced or completely abrogated binding (FIG. 2C). Three of thesix substitutions (L8G, G10R, and K11T) are located in Region 1, a loopthat contacts the Skp1-Fbw7 interface. The Arg-10 side-chain of the Ubvforms cation-pi interaction with the side-chain of Tyr-291^(Fbw7) andits aliphatic portion packs against the side-chains of Leu-288^(Fbw7)and Leu-116^(Skp1). Gly-8 and Thr-11 pack against Asn-108^(Skp1) and theside-chain NH₂ of Asn-108^(Skp1) forms a hydrogen bond with theside-chain OH of Thr-11 (FIG. 2B). The other three substitutions (R42I,H68R, and L73F) contact Skp1 only. The Ile-42 side-chain engages inhydrophobic interactions with the side-chain of Leu-34^(Skp1) and thePhe-73 side-chain packs against Pro-46^(Skp1) and Pro-48^(Skp1). TheArg-68 side-chain forms cation-pi interaction with the side-chain ofTyr-1 09^(Skp1) and polar contacts with the side-chains of Thr-26^(Skp1)and Asp-111^(Skp1) (FIG. 2B).

Notably, the surface on Skp1tr-F-box^(Fbw7) for binding to Ubv.Fw7.1largely overlaps with the previously elucidated surface on the analogousSkp1-F-box^(Fbl1) complex for binding to Cul1 (Zheng, 2002) (FIGS. 2 Dand E). To compare the energetics of Ubv.Fw7.1 and Cul1 binding toSkp1tr-F-box^(Fbw7), we constructed a series of point mutants atpositions within the common interface and measured the effects onbinding to both ligands (FIG. 2F). Three of the substitutions(N108A^(Skp1), Y109A^(Skp1) and D111R^(Skp1)), which reside in thecenter of binding surface, either abolished or significantly disruptedbinding to both Ubv.Fw7.1 and Cul1 and most of the other substitutionsalso had significant effects on binding to both ligands. These resultsshow that Ubv.Fw7.1 and Cul1 share a common structural and functionalbinding site on the Skp1 tr-F-box^(Fbw7) complex.

To confirm that Ubv.Fw7.1 and Cul1 target overlapping sites on theSkp1-Fbw7 complex, we tested whether Ubv.Fw7.1 can inhibit Cul1 bindingand SCF^(Fbw7) ligase activity. Cul1 has been reported to bind toSkp1-Fbw7 in vitro with picomolar affinity (Pierce, 2013). With surfaceplasmon resonance (SPR) analysis, we confirmed this tight interactionbetween Cul1 and Skp1-F-box^(Fbw7) (FIG. 6B) but we found that theinteraction with Skp1tr-F-box^(Fbw7) was ˜1000-fold weaker (FIGS. 6 A, Cand D). Thus, we used in vitro assays with Skp1tr-Fbw7 to show thatUbv.Fw7.1 inhibits the polyubiquitination activity of SCF^(Fbw7) (FIG.6E) and Cul1 binding (FIG. 6F). We speculated that this mode ofinhibition could be applied to other SCF ligases, prompting us tofurther characterize Ubv.Fw7.1 binding parameters with the ultimate goalof targeting other F-box proteins through the same mechanism.

Optimization of Ubvs for Binding to the Skp1-Fbw7 Complex

Ubv.Fw7.1 was selected for binding to a Skp1tr-Fbw7 complex thatcontained a truncated form of Skp1 optimized for structural analysis.However, our ultimate goal was to develop inhibitors of endogenous SCFligases, and Ubv.Fw7.1 bound only weakly to the Skp1-F-box^(Fbw7)complex containing full-length Skp1 (FIG. 3A), presumably due tounfavorable interactions with a negatively-charged loop near theN-terminus of Skp1 (FIGS. 6 A, G and H). To engineer Ubvs with enhancedaffinity for the Skp1-F-box^(Fbw7) complex, we designed asecond-generation library (Library 2) based on the sequence ofUbv.Fw7.1. Three residues involved in favorable contacts were heldconstant (Gly-8, Arg-10, Thr-11) while the remaining residues in contactwith the Skp1tr-F-box^(Fbw7) complex were “soft randomized” using amutagenesis strategy that favored the parental sequence but allowed foran 50% mutation frequency (FIG. 5). Following selections for binding tothe Skp1-F-box^(Fbw7) complex, 14 unique Ubvs were purified and ELISAsshowed dramatically improved affinities in comparison with Ubv.Fw7.1(FIG. 3A).

Many of the improved variants shared an A12G substitution and apreference for Arg at positions 49 and 75, and some also shared an I42Rsubstitution (FIG. 3A). While preference for Gly at position 12 isprobably due to optimization of Ubv interaction with the Skp1-Fbw7interface, Arg substitutions at positions 42, 49 and 75 can berationalized by the presence of a negatively-charged loop in full-lengthSkp1, which should come in contact with residues at these positions andwould thus favor the accumulation of positive charge in the Ubvs (FIG.6H). Ubv.Fw7.5, the tightest binder to Skp1-F-box^(Fbw7), exhibited anIC₅₀ of 45 nM and we focused on this variant for furthercharacterization.

Ubv.Fw7.1 and its relatives bind to the Skp1-Fbw7 complex mainly throughcontacts with Skp1, raising the possibility that these Ubvs may exhibitcross-reactivity with at least some of the many different humanSkp1-F-box complexes. Thus, we tested the binding of Ubv.Fw7.5 to sixSkp1-F-box domain complexes and, compared with Fbw7, we observed weakerbut significant binding to three of these (Fbw2, Fbl1 and Fbw5). Theaffinities correlated with the degree of sequence similarity with theFbw7 Ubv-binding region (FIG. 3B). Fbw2, which shares the highesthomology with Fbw7 exhibited an 8-fold lower affinity, while Fbw5 whichshows the least homology exhibited more than 50-fold lower affinity. Thethree F-box domains that did not bind to Ubv.Fw7.5 (Fbw1, Fbw11, andFbw12) showed the least homology with Fbw7.

Structure-Based Selection of Ubvs that Bind Specifically to theSkp1-F-Box^(Fbw11) Complex

Since contacts with F-box^(Fbw7) are mediated entirely by the Region 1loop of Ubv.Fw7.1, we considered whether sequence and length diversityin this loop could be exploited to alter specificity in favor ofparticular Skp1-F-box complexes. To explore this possibility, wedesigned a phage-displayed library (Library 3) in which four residues inRegion 1 of Ubv.Fw7.5 were replaced by completely random sequencesranging from 11 to 13 residues in length to increase the size of thepotential interaction interface with the F-box domain (FIG. 5). Library3 was selected for binding to the Skp1-F-box^(Fbw11) complex todetermine whether this approach could be used to alter the F-box domainpreference of Ubv.Fw7.5. Sequencing of 44 binding clones revealed that42 were identical and contained a 12-residue insertion in Region 1 (FIG.3C, Ubv.Fw11.1). Remarkably, purified Ubv.Fw11.1 protein was highlyspecific for Skp1-F-box^(Fbw11), as it bound very weakly to Skp1 incomplex with homologue F-box^(Fbw1) (89% sequence identity) and did notbind detectably to any of the other five Skp1-F-box complexes that wetested (FIG. 3B). To further improve affinity, we designed a library(Library 4) in which Region 1 of Ubv.Fw11.1 was soft randomized, andbinding selections yielded 16 unique Ubvs containing one to threesubstitutions (FIG. 7). Four of these variants exhibited enhancedaffinities for the Skp1-F-box^(Fbw11) complex (FIG. 3C) and the best ofthese (Ubv.Fw11.2) retained high specificity (FIG. 3B).

Intracellular Activity of Ubvs Targeting Fbw7 and Fbw11 Complexes

We transiently expressed Ubv.Fw7.5 or Ubv.Fw11.2 in HEK293T cells toascertain whether these Ubvs were able to exert effects in live cells.Since Fbw7 and Fbw11 protein complexes function as dimers (Suzuki, 2000;Welcker, 2013), expression vectors were designed to express Ubvs eitheras monomers or as dimers held together by a homodimeric GCN4 leucinezipper to enhance effective affinities through avidity (Table 2)(Harbury, 1993). To examine the interactions of Ubvs with endogenousproteins, Ubvs were immunoprecipitated, and co-precipitated proteinswere identified by mass spectrometry (FIG. 4A).

Consistent with the in vitro specificity profiles (FIG. 3B), Ubv.Fw7.5co-immunoprecipitated Fbw7 and Skp1, and also several other F-boxproteins including Fbw2 and Fbl1. Fbw7 was detected with the lowestspectral counts among the F-box proteins, but this is likely due to lowexpression levels of endogenous Fbw7. In support of this, a significantamount of Fbw7, but not Fbl1, co-immunoprecipitated with Ubv.Fw7.5 incells over-expressing Fbw7 or Fbl1 (FIG. 8A). In contrast, Ubv.Fw11.2was very specific for Fbw11, co-immunoprecipitating only Skp1, Fbw11 andsmall amounts of Fbw1. Similar levels of interacting proteins weredetected whether Ubvs were expressed as monomers or dimers, but Ubvdimers co-immunoprecipitated more non-specific proteins involved in cellhousekeeping functions (Table 4).

To determine whether Ubvs are able to disrupt interactions between Cul1and Skp1-F-box complexes in cells, exogenously expressed Fbw7 or Fbw11was immunoprecipitated in the absence or presence of Ubv. Expression ofUbv.Fw7.5 monomer or dimer significantly reduced or completely abrogatedthe co-immunoprecipitation of Cul1 with Fbw7, respectively, but did notaffect co-immunoprecipitation of Skp1 (FIG. 4B). In the case ofUbv.Fw11.2, expression of the dimer, but not the monomer, causedsignificant reduction in the amount of Cul1 (but not Skp1) thatco-immunoprecipitated with Fbw11, and this was consistent with the factthat the dimer but not the monomer co-immunoprecipitated with Fbw11(FIG. 4C). Thus, co-immunoprecipitation assays show that both Ubv.Fw7.5and Ubv.Fw11.2 interfere with the interactions between Skp1-F-boxcomplexes and Cul1 in cells but do not affect interactions between Skp1and F-box proteins, although dimerization is required to observe thiseffect in the case of Ubv.Fw11.2.

In order to determine whether cellular expression of Ubv.Fw7.5 orUbv.Fw11.2 led to inhibition of their corresponding ligases, we analyzedthe stability of ligase substrates. Expression of Ubv.Fw7.5 in eithermonomeric or dimeric format increased protein levels and decreaseddegradation rate of the SCF^(Fbw7) substrates Cyclin E and c-Myc tolevels comparable with those observed upon expression of an siRNAtargeting Fbw7 but had no effect on substrates of other SCF ligases,demonstrating that the observed inhibition was specific (FIG. 4D, 8C).In the case of Ubv.Fw11.2, assays were performed in the presence of ansiRNA targeting Fbw1 to reduce levels of SCF^(Fbw)1 (FIG. 8B), whichshares substrates with SCF^(Fbw11) Expression of the Ubv.Fw11.2 dimerand monomer increased the abundance and decreased the degradation rateof the SCF^(Fbw11) substrates Cdc25A and Wee1, which was similar tostabilization observed upon expression of an siRNA targeting Fbw11 (FIG.4E). Expression of the Ubv.Fw11.2 monomer had a smaller effect,consistent with the dimer being much more effective than the monomer indisruption of the interaction between Cul1 and the Skp1-Fbw11 complex(FIG. 4C). The inhibitory effect of Ubv.Fw11.2 was specific toSCF^(Fbw11), as it did not affect substrates of other SCF ligases (FIG.8D) and it did not stabilize substrates of Fbw1/11 in the background ofFbw11 siRNA treatment (FIG. 8E). Since Fbw7 and Fbw11 are involved incell cycle progression (Wang, 2014), we also tested whether inhibitionof these E3 ligases by Ubvs exerts any effect on cell cycle. While wedid not detect any large effects, the small changes that were observed(decrease in G1 population for Ubv.Fw7.5 and increase in G2/M populationfor Ubv.Fw11.2) (FIGS. 8 F and G) were similar to those obtained withsiRNA treatment and consistent with the previously reported effects ofFbw7 (Wu, 2015) and Fbw11 inhibition (Guardavaccaro, 2003). Takentogether, these data show that engineered Ubvs interact with endogenousSkp1-F-box complexes in cells and cause displacement of Cul1 andconsequent inhibition of specific SCF E3 ligases.

Materials and Methods

Protein Expression and Purification.

His-tagged Fbw7 and GST-tagged Skp1 were co-expressed from dicistronicmRNA. Ubvs and Cul1N-terminal domain (NTD) were expressed with His-FLAGtag. See Table 2 for detailed list of all expression constructs. Allproteins were expressed in Escherichia coli BL21 (pLys) cells, whichwere grown to OD₆₀₀ 0.6-1.0 and induced with 1 mM IPTG either overnightat 16° C. (for Skp1-F-box complexes and Cul1 NTD) or for 3 hours at 37°C. (for Ubvs). Cells were lysed and proteins were purified by Ni-NTAchromatography using standard techniques. Since expression of Skp1 washigher than expression of F-box proteins, purifying Skp1-F-box complexesthrough His-tagged F-box proteins ensured that only complexes werepurified. Eluted proteins were dialyzed into 50 mM Hepes, pH 7.5, 500 mMNaCl, 10% glycerol, 1 mM DDT buffer and stored at 4° C. or frozen at−80° C. for further applications.

Phage-Displayed Ubv Library Design and Construction.

Library 1 in this study is the same as Library 2 in a previous study(Ernst). Libraries 2, 3 and 4 in this study were constructed usingmethods described previously (Fellouse, 2007). For the construction ofLibrary 2, a phagemid designed for the phage display of Ub (Ernst) wassubjected to site-directed mutagenesis with degenerate oligonucleotidesto simultaneously mutate three regions in the gene encoding for Ub.Positions were diversified with a “soft randomization” strategy (Sidhu,2000), in which the nucleotide ratio at degenerate positions wasadjusted to 70% of the wt nucleotide and 10% of each of the othernucleotides. See FIG. 5 for original sequence and positions targeted fordiversification, and Table 5 for oligonucleotides used for libraryconstruction. For the construction of Libraries 3 and 4, a phagemid wasdesigned for the display of an Ub variant in which positions 1-35 werewt sequence and positions 42-76 were the sequence of Ubv.Fw7.5. For theconstruction of Library 3, a set of mutagenic oligonucleotides was usedto replace Ub positions 8-11 with completely random sequences containing11-13 residues (FIG. 5, Table 5). For the construction of Library 4, amutagenic oligonucleotide was used to replace positions 8-11 with asoft-randomized sequence corresponding to the sequence of Ubv.Fw11.1(FIG. 5, Table 5). The diversities of the constructed libraries were asfollows: Library 2, 2.2×10⁹; Library 3, 5.0×10⁹; Library 4, 1.5×10⁹.

Selection of Ubv Variants.

GST-tagged target proteins (GST-Skp1:His-F-box) were coated on 96-wellMaxiSorp plates (Thermoscientific 12565135) by adding 100 μL of 1 μMproteins and incubating overnight at 4° C. Five rounds of bindingselections with phage library pools were performed against immobilizedproteins as described (Fellouse, 2007). To eliminate Ubv-phage thatbound nonspecifically, input phage pools were either mixed withnon-target proteins (round 1) or pre-incubated on plates coated withnon-target proteins (rounds 2-5). The non-target proteins were GST forselections with Libraries 1 and 2 or a mix of non-target Skp1-F-boxcomplexes for selections with Libraries 3 and 4.

ELISAs.

GST-tagged target proteins were immobilized on 384-well MaxiSorp plates(Thermoscientific 12665347) by adding 30 μL of 1 μM proteins forovernight incubation at 4° C. or for 2 hour incubation at roomtemperature. Phage and protein ELISA against immobilized proteins wereperformed as described (Fellouse, 2007), except that three washes wereperformed for all wash steps and volumes were scaled down from 100 μL to30 μL to accommodate the 384-well format. Binding of phage was detectedusing anti-M13-HRP antibody (1:5000 dilution, GE Healthcare 27-9421-01)and binding of FLAG-tagged ligands (Ubv or Cul1) was detected usinganti-FLAG-HRP antibody (1:5000 dilution, Sigma A8592). To measureprotein ELISA EC₅₀ values, the concentration of ligand proteins (Ubv orCul1) was varied, while the concentration of target proteins(GST-Skp1:His-F-box) immobilized on the plate remained constant. EC₅₀values were calculated by fitting the obtained binding curves to fourparameter logistic non-linear regression model and corresponded toligand concentration (curve inflection point) at which 50% of bindingwas observed. To measure protein ELISA IC₅₀ values, the concentration oftarget in solution (Skp1-F-box) was varied, while the concentration oftarget immobilized on the plate (GST-Skp1:His-F-box) and concentrationof ligand (Ubv) in solution remained constant. IC₅₀ values, whichcorresponded to the concentration of target in solution which inhibited50% of ligand binding to the immobilized target, were calculated byfitting the data as described for EC₅₀ values.

Surface Plasmon Resonance Analysis.

SPR measurements were performed at 25° C. using ProteOn XPR36 instrument(Bio-Rad). Skp1tr-F-box^(Fbw7) and Skp1-F-box^(Fbw7) ligands wereimmobilized by amine coupling to GLC sensor chip surface. Cul1 NTD wasdiluted into PBT buffer (phosphate buffered saline, 0.05% Tween, and0.5% BSA) and injected for 360 sec at 50 μL/min. Dissociation wasmonitored for 1200 sec in PBT buffer. Sensorgrams were fitted to 1:1Langmuir model using ProteOn Manager Software (Bio-Rad).

Protein Purification for Crystallization and Structure Determination.

Complex consisting of GST-tagged Skp1tr and His-tagged F-box^(Fbw7) waspurified on Ni-NTA resin from cells co-expressing both proteins (seeTable 2 for constructs used). To remove GST and His purification tags(containing TEV protease cleavage sites) the obtained complex was firstbound to glutathionine resin, next eluted from the resin by TEV cleavageof GST tag and finally re-purified on Ni-NTA resin to remove His-taggedTEV protease and other impurities. Similarly, His-tagged Ubv.Fw7.1 waspurified on Ni-NTA resin, His tag (containing TEV protease cleavagesite) was removed by TEV protease and cleaved Ubv was re-purified onNi-NTA resin. Cleaved Skp1tr-F-box^(Fbw7) complex was mixed with excessof cleaved Ubv.Fw7.1 and subjected to gel filtration chromatography. Asingle peak corresponding to Skp1tr-F-box^(Fbw7)-Ubv.Fw7.1 complex wascollected, exchanged into 20 mM Hepes pH 7.5, 200 mM NaCl, 1 mM DTTbuffer, and concentrated to 19 mg/ml. Crystals were grown by mixingequal volumes of Skp1-F-box^(Fbw7)-Ubv.Fw7.1 solution with the reservoirsolution (100 mM acetate pH 4.5, 12% PEG 4000, 15% glycerol) andincubating at 20° C. The crystals were cryoprotected by soaking inreservoir solution with a final glycerol concentration of 20%. Data wascollected at NE-CAT 24 ID-C (APS, Chicago, Ill.) and processed withHKL2000 (Otwinowski, 1997). The structure was solved by molecularreplacement with Phaser (McCoy, 2007) using structures of Skp1tr-Fbw7(PDB:2OVR) and ubiquitin (PDB:1 UBQ) as search models. The model wasrebuilt using Coot (Emsley, 2004) and refined to 2.5 Å with a workingR_(value) of 20.0% and R_(free) of 25.0% using PHENIX (Adams, 2010).

Protein Expression Constructs Used in Cell-Based Assays.

Genes encoding for FLAG-tagged Ubvs were cloned into pcDNA3.1/nFLAG-Destvector for monomer expression or into the same vector modified to encodea GCN4 leucine zipper dimerization sequence(RMKQLEDKIEELLSKIYHLENEIARLKKLIGER) inserted in place of vectornucleotides 944-976) for dimer expression. Cul1, Fbw11, Fbw1 and Fbw7were expressed from pcDNA3.1 based vectors (See Table 2 for additionaldetails).

Cell-Based Functional Assays.

On day 0, 6-well plates were seeded with 4×10⁵ HEK293T cells. On day 1,cells were transfected with 2 μg of plasmid DNA (empty vector, variousFLAG-Ubv or various FLAG-F-box constructs) using the X-tremeGENEtransfection reagent (Roche 06365809001), according to manufacturer'sprotocol. After 6 hours, media was removed and replaced with freshmedia, and cells were subjected to a second round of transfection with10 nM siRNA (Control, Fbw1, Fbw11, Fbw1+Fbw11 or Fbw7) usingLipofectamine RNAiMAX (Invitrogen 13778-075), according tomanufacturer's instructions. siRNA included the following: SilencerSelect Negative Control #1 (Invitrogen 4390843); Fbw1:CGGAAGAGUUUUUCGACUAtt (Invitrogen 17110); Fbw11: GGUUGUUAGUGGAUCAUCAtt(Invitrogen s23485); Fbw7: CGGGUGAAUUUAUUCGAAAtt (Invitrogen s30665).

On day 2, media was replaced with fresh media. On day 3, cycloheximide(100 μg/ml) was added for 0-6 hours. Cells were lysed in lysis buffer(Cell Signalling 9803) and cell lysates were subjected to western blotanalysis with antibodies against endogenous proteins (Cdc25A (Upstate05-743), Wee1 (Cell Signaling 4936), c-Myc (Santa Cruz SC-40), Cyclin E(ABCAM 3927), p27 (BD Transduction Laborotories 610241), Cry2 (Abcam93802), GADPH (Cell Signaling 2118L) or FLAG-tagged proteins(Sigma-Aldrich A8592)).

Flow Cytometry Analysis.

HEK293T cells were treated as described in Cell-based functional assayssection. Two days post transfection, cells were re-suspended inphosphate buffered saline (PBS), fixed by addition of 70% ethanol andstored at −20° C. Immediately prior to flow cytometry analysis fixedcells were washed in PBS, re-suspended in 500 μL PBS at concentration of1×10⁶ cells/ml, and stained by addition of Hoechst 33342 (LifeTechnologies H3570) dye to final concentration of 4 μg/ml. Stained cellswere analyzed by UV excitation at 355 nm on a BD LSRFortessa X-20 cellanalyzer and detected using a 450/50 nm bandpass filter. The dataacquired was analyzed by FlowJo10 software.

Immunoprecipitation Assays.

HEK293T cells were grown to 70-80% confluency on 10-cm plates andtransfected with 10 μg total plasmid DNA (HA-F-box, FLAG-Ubv,FLAG-Cullin1, empty vector or various combinations) using theX-tremeGENE 9 transfection reagent (Roche 06365809001) according tomanufactures instructions. Cells were harvested 2 days post transfectionand cell pellets were frozen for further applications. Forco-immunoprecipitation analysis, cells were re-suspended in 1 ml lysisbuffer (25 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5%glycerol, protease inhibitor cocktail (Sigma S8830)), and after 1 hincubation at 4° C., cell lysate was clarified by centrifugation at15000 rpm for 10 min. 800 μg of total cell lysate was incubated with 3μg of anti-FLAG (Sigma F1804) or anti-HA (Sigma 3663) antibody overnightat 4° C. Immunoprecipitations were performed using Protein A/G Agarose(Thermo Scientific™ Pierce 20422) according to manufacturer's protocol.Immunoprecipitated proteins were visualized by western blot usinganti-FLAG-HRP (Sigma A8592), anti-HA-HRP (Sigma H6533), and anti-Skp1(Abcam 10536) antibodies.

Mass Spectrometry Analysis.

Frozen cell pellets were thawed into 1 ml High Salt AFC buffer (10 mMTris-HCl pH7.9, 420 mM NaCl, 0.1% NP-40, 1 mM sodium orthovanadate, 2 mMsodium pyrophosphate, 10 mM NaF, protease inhibitor cocktail (SigmaS8830)). Cell suspensions were then were subjected to 3 freeze-thawcycles, sonicated (5 cycles of 0.3 sec on and 0.7 sec off) and incubatedfor 30 min at 4° C. with 12.5-25 U of benzonase nuclease (Sigma E1014).The samples were centrifuged at 13,000 rpm for 30 min at 4° C. and 10 μlslurry of M2 anti-Flag beads (Sigma 8823) was added for overnightincubation. Beads were washed 2 times with low salt AFC buffer (10 mMTris-HCL, pH7.9, 100 mM NaCl, 0.1% NP-40) and 3 times with low salt AFCbuffer without detergent. Immunoprecipitated proteins were eluted fromthe beads with 0.5 M ammonium hydroxide, 4×50 μl for a total of 200 μl.Samples were snap frozen in liquid nitrogen and dried. Furtherpreparation of samples, mass spectrometry and analysis of obtained datawas performed as previously described (Marcon, 2014).

TABLE 2 Protein expression vectors Constructs Protein residues^(a)Vector N-terminus Tag residues^(b) Bacterial vectors Fbw7 (F-box-WD40)263-708 pPROEX His-TEV MSYYHHHHHHDYDIPTTENLYF*QGA Fbw7 F-box 263-367 HTbFbw7 F-box (Structure) 263-323 Fbw2 F-box 53-104 Fbw5 F-box 1-79Fbl1 F-box 95-139 Fbw12 F-box 1-49 Fbw1 F-box 101-214 Fbw11 F-box 80-191Skp1 Full length pPROEX GST-TEV GST-DYDIPTTENLYFQ*GA Skp1 (Loop 1Δ)38-43Δ HTb 70-77Δ, Skp1 (Loop 2Δ) K78G E79S K80G R81G Skp1trK78G E79S K80G (ELISA/Structure) R81G Cul1 NTD^(c) 1-410 P11 His-TEV-MGSSHHHHHHSSGRENLYFQ*GHMDY (V367D L371E) FLAG KDDDDKUbv.Fw7.1 (Structure) 1-76 p53DEST His-TEV MAHHHHHHVTSLYKKAGENLYFQ*GS GSUbv (ELISA) Full length p53DEST His-FLAG MAHHHHHHVTSLYKKAGDYKDDDDKMammalian vectors FLAG-Ubv Full length pcDNA3.1/ FLAG-MDYKDDDKGQGPDPSTNSADITSLYK monomer nFLAGDEST Linker- KAGTMDYKDDDDK FLAGFLAG-Ubv Full length pcDNA3.1/ FLAG- MDYKDDDKGQRMKQLEDKIEELLSKILeu-zipper dimer nFLAGDEST + Leu Zipper- YHLENEIARLKKLIGERTSLYKKAGTLeu-zipper) FLAG MDYKDDDDK HA-Fbw7 Full length pcDNA3.1/ HAMYPYDVPDYAGQGPDPSTNSADITSL HA-Fbw11 (IsoformB) nHADEST YKKAGST FLAG-Cul1Full length pcDNA3.1/ FLAG MDYKDDDDKGQGPDPSTNSADITSLY FLAG-Fbw7nFLAGDEST KKAGT FLAG-Fbw11(Isoform B) FLAG-Fbw1 Full length pcDNA3 FLAGMDYKDDDDK ^(a)Residue limits of canonical human isoforms. Any amino acidsubstitutions relative to wt are indicated if present. ^(b)Tag residuesare shown, except for GST tag. Residues that comprise functionalelements are underlined and TEV cleavage site denotedby *. All tags areN-terminal. ^(c)Cul1 N-terminal domain (NTD) was expressed with V376Dand L317E substitutions that are necessary for solubility in the absenceof the C-terminal domain as described (Zheng, 2002).

TABLE 3 Data collection and refinement statistics for Skp1tr-F-box^(Fbw7)-Ubv.Fw7.1 Complex Data collection Space group P2₁2₁2₁ Celldimensions a, b, c (Å) 63.4, 98.0, 107.7 α, β, γ (°) 90, 90, 90Resolution (Å) 50.0-2.5 (2.56-2.50) ^(a) R_(meas) 0.052 (0.359) I/σI19.6 (1.3) Completeness (%) 99.4 (99.1) Redundancy 4.4 (3.9) RefinementResolution (Å) 50.0-2.5 (2.56-2.50) No. reflections 23612 (1532)R_(work)/R_(free) 20.0/24.0 (30.9/39.4) No. atoms Protein 4246 Water 38B-factors Protein 81.1 Water 51.2 R.m.s. deviations Bond lengths (Å)0.004 Bond angles (°) 0.879 Ramachandran favored 97.6 allowed 2.4outliers 0 ^(a) Values in parenthesis correspond to the highest shell.

TABLE 4 Mass spectrometry analysis of Ubv interactions in HEK293T cellsFw7.5 Fw11.2 ID^(a) Gene^(b) Description V^(c) Ub^(c) Fw7.5 dimer Fw11.2dimer All Ubv P63208 SKP1 Skp1  0^(d) 0 18 37 57 40 P68363 TUBA1BTubulin 21  12 21 66 36 2 Q09028 RBBP4 Histone binding 2 0 0 12 0 0P53671 LIMK2 Histone kinase 0 0 10 10 8 5 Q9H479 FN3KKetosamine-3-kinase 0 0 9 0 4 0 O15018 PDZD2 PDZ domain 0 0 2 5 5 1containing Q8NI27 THOC2 mRNA processing 0 0 5 5 0 3 Monomer Ubv specificO95479 H6PD Glucose 0 0 203 0 143 0 dehydrogenase Q9UIV8 PI13 Peptidaseinhibitor 0 0 146 0 120 0 Q96QC0 PPP1R10 Phosphatase 0 0 6 0 6 0 DimerUbv specific Q15233 NONO mRNA processing 0 0 0 118 3 114 Q8WXF1 PSPC1mRNA processing 3 0 0 56 0 51 P23246 SFPQ mRNA processing 5 2 0 60 2 70Q15691 MAPRE1 Microtubule binding 0 0 0 58 0 48 P17980 PSMC3 26Sproteasome 0 0 0 39 0 37 subunit Q9P2E9 RRBP1 Ribosome receptor 0 0 0 130 28 Q9Y490 TLN1 Cytoskeletal 0 0 2 38 0 10 component P42566 EPS15 EGFRsubstrate 0 0 0 20 0 27 Q9UII2 ATPIF1 ATPase inhibitor 0 0 0 27 0 19O00233 PSMD9 26S proteasome 0 0 0 14 0 21 subunit P05787 KRT8 Keratin 00 0 20 0 18 Q16204 CCDC6 Unknown 0 0 0 18 0 13 Q15019 SEPT2 CytoskeletalGTPase 0 0 0 15 0 10 Q14203 DCTN1 Microtubule transport 0 0 0 9 0 17P46736 BRCC3 Lys-63 deubiquitinase 0 0 0 15 0 16 P80303 NUCB2 Calciumhomeostasis 0 0 0 8 0 14 Q07065 CKAP4 Cytoskeleton 0 0 0 14 0 12associated P31146 CORO1A Cytoskeletal 0 0 0 12 0 7 component O60271SPAG9 Scaffold protein 0 0 0 9 0 13 Q9BW19 KIFC1 Microtubule transport 00 0 5 0 13 P16220 CREB1 Transcription factor 0 0 0 8 0 10 Q13625 TP53BP2p53 regulator 0 0 0 5 0 8 O00139 KIF2A Microtubule transport 0 0 0 9 0 7O43293 DAPK3 Serine/Threonine 0 0 0 9 0 6 kinase Q9NVA2 SEPT11Cytoskeletal GTPase 0 0 0 8 0 9 Q13976 PRKG1 Serine/Threonine 0 0 0 7 09 kinase Q14141 SEPT6 Cytoskeletal GTPase 0 0 0 4 0 8 Q9P0K7 RAI14 Actinassociated 0 0 0 2 0 7 Q16181 SEPT7 Cytoskeletal GTPase 0 0 0 10 0 10Q14980 NUMA1 Nuclear matrix 0 0 0 4 0 10 Q96CN9 GCC1 Golgi associated 00 0 4 0 7 P40222 TXLNA Vesicle traffic 0 0 0 3 0 7 095396 MOCS3 tRNAbiosynthesis 0 0 0 2 0 7 Q9UPY8 MAPRE3 Microtubule 0 0 0 2 0 7associated P62195 PSMC5 26S proteasome 0 0 0 6 0 3 subunit Q8N302 AGGF1Angiogenic factor 0 0 0 2 0 6 Q15390 MTFR1 Mitochondrial fission 0 0 0 00 6 O75146 HIP1R Clathrin associated 0 0 0 5 0 3 P53621 COPA Golgi-to-ERtransport 0 0 0 5 0 2 O60308 KIAA0562 Centrosomal protein 0 0 0 5 0 2Q969V6 MKL1 Transcription factor 0 0 0 4 0 5 Q15007 WTAP mRNA processing0 0 0 2 0 5 Q8TBA6 GOLGA5 Golgi formation 0 0 0 2 0 5 Q4VCS5 AMOT Tightjunction 0 0 0 0 0 5 maintenance Q9H6D7 HAUS4 Spindle assembly 0 0 0 0 05 Ubv.Fw11.2 specific Q9UKB1 FBXW11 F-box 0 0 0 0 125 85 Q9Y297 FBXW1F-box 0 0 0 0 10 4 P0C0L4 C4A Complement comp. 0 0 0 0 13 3 Q06203 PPATRibosyl transferase 0 0 0 0 13 3 P04632 CAPNS1 Protease subunit 0 0 0 012 0 Q96K76 USP47^(e) Ubiquitin protease 0 0 0 0 3 7 Q01664 TFAP4Transcription factor 0 0 0 0 6 0 Ubv.Fw7.5 specific Q9UKT8 FBXW2 F-box 00 24 18 0 0 P19838 NFKB1 Transcription Factor 0 0 22 0 0 0 Q13309 SKP2F-box 0 0 12 7 0 0 Q7Z6M2 FBXO33 F-box 0 0 3 7 0 0 Q9NVF7 FBXO28 F-box 00 0 7 0 0 Q969H0 FBXW7 F-box 0 0 5 0 0 0 O94952 FBXO21 F-box 0 0 3 5 0 0^(a)ID and ^(b)Gene name for identified proteins showing >2-foldenrichment relative to control and 5 peptides or more in any of the Ubvsamples. ^(c)Controls corresponds to HEK293T cells transfected withempty vector (V) or Ub Δ75G76G (Ub). ^(d)Numbers of endogenous peptidescorresponding to the identified protein. ^(e)USP47 is a known interactorof Fbw1 and Fbw11(Peschiaroli, 2010).

TABLE 5 Oligonucleotides used for construction of Ubv Libraries OligoSequence^(a) Library oMg210 ATG CAG ATT TTC GTG (5)(5)(5) 2(5)(6)(6) GGT (7)(6)(5) CGT ACC (7)(6)(5) ATC ACC CTC GAG GT oMG212AAG ATC CAG GAT AAG (7)(5)(5) GGA 2 ATT CCT CCT GAT CAG CAG (5)(8)(8)CTG (5)(1)(1) TTT (8)(6)(6) (6)(7)(8) (5)(5)(7) (6)(8)(6) CTGGAA GAT GGA CGT oMG214 ATT CAA AAG GAG TCT (5)(6)(8) CTT 2(6)(7)(8) CTT (7)(8)(7) (8)(8)(7) (N5)(8)(8) (8)(8)(8) (6)(7)(8)(7)(7)(8) (7)(7)(8) GGC GGT GGC GGA TCC oMG281ATT TTC GTG AAA ACCNNK NNK NNK NNK 3 NNK NNK NNK NNK NNK NNK NNK ACCATC ACC CTC GAG oMG282 ATT TTC GTG AAA ACC NNK NNK NNK NNK 3NNK NNK NNK NNK NNK NNK NNK NNK ACC ATC ACC CTC GAG oMG283ATT TTC GTG AAA ACC NNK NNK NNK NNK 3NNK NNK NNK NNK NNK NNK NNK NNK NNK ACC ATC ACC CTC GAG oMG289ATTTTCGTGAAAACC (8)(5)(9) (6)(6)(6) 4 (8)(5)(9) (5)(5)(5) (8)(6)(9)(7)(7)(8) (8)(6)(9) (8)(5)(9) (6)(5)(9) (5)(5)(9) (8)(5)(9)ACCATCACCCTCGAG ^(a)Numbers denote specific nucleotide mixtures: 5 = 70%A and 10% other nucleotides; 6 = 70% C and 10% other nucleotides; 7= 70% G and 10% other nucleotides; 8 =  70% T and 10% other nucleotides;9 = 90% T and 10% G. “N” denotes and equimolar mixture of all fournucleotides. “K” denotes an equimolar mixture of G and T.

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What is claimed is:
 1. A ubiquitin variant (Ubv) polypeptide comprisingone or more amino acid substitution in one or more region of a ubiquitinpolypeptide (SEQ ID NO:1), wherein the region is selected from the groupconsisting of: (a) region 1 (amino acids 2-14 of SEQ ID NO:1) whereinthe polypeptide comprises the structure:X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄ wherein X₂ is selected fromthe group consisting of H and Q; X₄ is selected from the groupconsisting of F and L; X₈ is selected from the group consisting of G andL; X₉ is selected from the group consisting of A, S, and T; X₁₀ isselected from the group consisting of G, R, and V; X₁₁ is selected fromthe group consisting of K and T; X₁₂ is selected from the groupconsisting of A, G, N, and T; X₁₄ is selected from the group consistingof I and T; any X not specified optionally has the amino acid sequenceof the corresponding position in SEQ ID NO:1; and the polypeptideoptionally comprising 1-4 additional amino acids; (b) region 2 (aminoacids 42-49 of SEQ ID NO:1) wherein the polypeptide comprises thestructure: X₄₂-X₄₃-X₄₄-X-₄₅-X₄₆-X₄₇-X₄₈-X₄₉ wherein X₄₂ is selected fromthe group consisting of I, R, T, and V; X₄₄ is selected from the groupconsisting of I, L, and V; X₄₆ is selected from the group consisting ofA, R, S, and Y; X₄₇ is selected from the group consisting of G, H, K,and R; X₄₈ is selected from the group consisting of K, M, and R; X₄₉ isselected from the group consisting of K, L, P, Q, R, and V; and any Xnot specified optionally has the amino acid sequence of thecorresponding position in SEQ ID NO:1; and (c) region 3 (amino acids62-78 of SEQ ID NO:1) wherein the polypeptide comprises the structure:X₆₂-X₆₃-X₆₄-X₆₅-X₆₆-X₆₇-X₆₈-X₆₉-X₇₀-X₇₁-X₇₂-X₇₃-X₇₄-X₇₅-X₇₆-X₇₇-X₇₈wherein X₆₂ is selected from the group consisting of E, H, and Q; X₆₃ isselected from the group consisting of K and R; X₆₆ is selected from thegroup consisting of S and T; X₆₈ is selected from the group consistingof H, Q, and R; X₇₀ is selected from the group consisting of L and V;X₇₁ is selected from the group consisting of L, M, V, W, and Y; X₇₂ isselected from the group consisting of F, I, L, R, and V; X₇₃ is selectedfrom the group consisting of F, L, and V; X₇₄ is selected from the groupconsisting of G, L, P, R, and S; X₇₅ is selected from F, G, K, R, and S;X₇₆ is selected from the group consisting of A, E, G, L, N, R, and V;X₇₇ is selected from the group consisting of E, G, R, and T, or isabsent; X₇₈ is selected from the group consisting of A, G, P, and S, oris absent; and wherein X₃₄ is selected from D, E, and G, and any X notspecified in said Ubv polypeptide optionally has the amino acid sequenceof the corresponding position in SEQ ID NO:1; or a fragment thereof,wherein the sequence of said Ubv polypeptide does not consist of SEQ IDNO:1.
 2. The Ubv polypeptide of claim 1, comprising a substitution inone or more position selected from the group consisting of X₈, X₁₁, andX₇₃ of the amino acid sequence of SEQ ID NO:1.
 3. The Ubv polypeptide ofclaim 2, comprising one or more substitution selected from L8G, K11T,and L73F.
 4. The Ubv polypeptide of claim 2 or 3, further comprising asubstitution in position X₄₂ and/or X₆₈.
 5. The Ubv polypeptide of claim4, wherein said substitution in position X₄₂ and/or X₆₈ is R42I and/orH68R.
 6. The Ubv polypeptide of any one of claims 2-5, comprising eachof said substitutions and further comprising an amino acid substitutionin one or more position selected from the group consisting of X₉, X₁₀,X₁₂, X₄₆, X₄₇, X₄₉, X₆₂, X₆₃, X₇₂, X₇₆, X₇₇, and X₇₈.
 7. The Ubvpolypeptide of claim 6, wherein said substitution in one or moreposition selected from the group consisting of X₉, X₁₀, X₁₂, X₄₆, X₄₇,X₄₉, X₆₂, X₆₃, X₇₂, X₇₆, X₇₇, and X₇₈, is one or more of the followingsubstitutions T9A, G10R, T12A, A46S, G47R, Q49L, Q62H, K63R, R72I, R76N,G77E, and G78S.
 8. The Ubv polypeptide of claim 7, comprising each ofsaid substitutions (Fw7.1).
 9. The Ubv polypeptide of claim 3,comprising each of said substitutions and the following substitutions:(a) G10V, T12N, T14I, R42T, Q62E, T66S, H68R, R74G, R76L, 77T, and 78A(Fw7.2); (b) Q2H, F4L, G10V, R42I, I44V, A46Y, H68Q, V70L, R74L, 77R,and 78P (Fw7.3); or (c) T9A, T12N, R42I, A46S, Q49L, Q62H, K63R, H68R,R72I, R76N, 77E, and 78A (Fw7.4).
 10. A Ubv polypeptide comprising oneor more amino acid substitution selected from the group consisting ofA12G, I42R or V, L49R, H62Q, R63K, and G75R in the amino acid sequenceof Fw7.1, and wherein amino acids 77 and 78 are optional.
 11. The Ubvpolypeptide of claim 10, comprising the following substitutions: A10G,I42V, L49R, H62Q, R63K, I72V, R74G, G75R, and G76R (Fw7.5), and whereinamino acids 77 and 78 are optional.
 12. The Ubv polypeptide of claim 10,comprising the following substitutions: (a) A10G, L49R, H62Q, R63K,R74P, and G75S (Fw7.6); (b) A10G, I42R, S46A, R47G, K48R, L49R, H62Q,R63K, L71Y, I72V, and G75R (Fw7.7); (c) I42R, S46A, R47G, H62Q, R63K,L71W, I72F, F73L, G75F, and G76V (Fw7.8); (d) A10G, L49R, H62Q, R63K,L71M, I72V, R74S, and G75K (Fw7.9); (e) A9T, A10G, I42V, I44L, L49V,H62Q, R63K, L71W, I72L, F73L, and G75R (Fw7.10); (f) E36G, I42V, R47K,L49R, H62Q, R63K, and G75R (Fw7.11); (g) I42R, S46A, R47G, L49R, H62Q,R63K, I72L, G75R, and G76E (Fw7.12); (h) A9S, A10G, E36G, I44L, L49R,H62Q, R63K, R74S, and G76A (Fw7.13); (i) E36D, I42V, I44L, S46R, L49K,H62Q, R63K, V70L, L71W, F73V, and G75R (Fw7.14); (j) A10G, R47H, K48M,L49R, H62Q, R63K, and R74S (Fw.7.15); (k) A10G, E36G, I44L, L49P, H62Q,R63K, L71V, I72V, and G75R (Fw7.16); (l) A10G, E36G, I42R, S46A, R47G,L49R, H62Q, R63K, and T66S (Fw7.17); and (m) I42R, L49R, H62Q, R63K,I72L, and R74P (Fw7.18); wherein amino acids 77 and 78 are optional. 13.A Ubv polypeptide comprising the amino acid sequence of Fw11.1 (SEQ IDNO:20), optionally comprising 1-5 substitutions.
 14. The Ubv polypeptideof claim 13, comprising the following substitutions: (a) S12Y, S14T, andN17H (Fw11.2; SEQ ID NO:21); (b) S14T and Y15F (Fw11.3; SEQ ID NO:22);(c) S12Y (Fw11.4; SEQ ID NO:23); (d) S14T (Fw11.5; SEQ ID NO:24); (e)S14T, Y15F, and N17D (Fw11.6; SEQ ID NO:25); (f) S14T and N17H (Fw11.7;SEQ ID NO:26); (g) S12Y and S14T (Fw11.8; SEQ ID NO:27); (h) Y15F(Fw11.9; SEQ ID NO:28); (i) N17H (Fw11.10; SEQ ID NO:29); (j) S14N andY15F (Fw11.11; SEQ ID NO:30); (k) N17D (Fw11.12; SEQ ID NO:31); (l) S12Hand N17Y (Fw11.13; SEQ ID NO:32); (m) S12Y and S15F (Fw11.14; SEQ IDNO:33); (n) K11R and S14T (Fw11.15; SEQ ID NO:34); (o) S12T (Fw11.16;SEQ ID NO:35); and (p) S12A (Fw11.17; SEQ ID NO:36).
 15. A UbVpolypeptide comprising a sequence selected from the group consisting ofSEQ ID NOs:2-36, or a variant thereof comprising a sequence that is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NOs:2-36, or a fragment thereof.
 16. A nucleic acid moleculeencoding a Ubv polypeptide of any one of claims 1-15.
 17. A recombinantexpression vector comprising a nucleic acid molecule of claim
 16. 18. Ahost cell comprising a nucleic acid molecule of claim 16 or arecombinant expression vector of claim
 17. 19. A method for obtaining aubiquitin variant polypeptide that binds to a Skp1-F-box proteincomplex, the method comprising randomizing sequences in region 1, region2, and/or region 3 of ubiquitin to create a library of variants, andscreening the library of variants for binding to the F-box protein ofsaid Skp1-F-box protein complex, or a fragment thereof, optionallywherein region 1 comprises up to 8 amino acids in addition to thosepresent in ubiquitin.
 20. The method of claim 19, wherein the F-boxprotein comprises Fbw7 or Fbw11, or a fragment thereof.
 21. A method ofmodulating the activity of an Skp1-Cul1-F-box (SCF) E3 ligase in a cell,the method comprising contacting the cell with an agent that altersbinding of Cul1 to a complex comprising Skp1 and an F-box protein in thecell.
 22. The method of claim 21, wherein said agent decreases theactivity of said SCFE3 ligase.
 23. The method of claim 20, wherein saidagent decreases or inhibits binding of Cul1 to said complex comprisingSkp1 and an F-box protein.
 24. The method of any one of claims 21-23,wherein said agent comprises a Ubv polypeptide, a nucleic acid moleculeencoding a Ubv polypeptide, or a fragment thereof.
 25. The method of anyone of claims 21-24, wherein said agent has specificity for a particularSCF E3 ligase.
 26. The method of any one of claims 21-24, wherein saidagent is active against more than one SCF E3 ligase.
 27. The method ofany one of claims 21-26, wherein said SCF E3 ligase comprises Fbw7 orFbw11.
 28. The method of claim 24, wherein said Ubv polypeptide is a Ubvpolypeptide of any one of claims 1 to 15, or said nucleic acid moleculeis a nucleic acid molecule of claim
 16. 29. The method of any one ofclaims 21-28, wherein said cell is a cancer cell.
 30. The method ofclaim 29, wherein said cancer cell is comprised within a subject havingcancer.
 31. A method of treating cancer in a subject, the methodcomprising modulating the activity of an Skp1-Cul1-F-box (SCF) E3 ligasein a cell of the subject according to the method of any one of claims21-30.
 32. A method of identifying an agent that modulates the activityof an SCF E3 ligase in a cell, the method comprising contacting a cellexpressing an SCF E3 ligase with a candidate agent, and determiningwhether said agent affects the binding of Cul1 to a complex comprisingSkp1 and an F-box protein.
 33. The method of claim 32, wherein said cellfurther expresses a Ubv polypeptide.
 34. The method of claim 32 or 33,wherein said candidate agent comprises a small molecule compound. 35.The method of any one of claims 32-34, wherein said determining iscarried out using an immunoprecipitation assay.
 36. The method of anyone of claims 33-35, wherein said Ubv polypeptide is a Ubv polypeptideof any one of claims 1-14.